Supply of regenerated nitrogen to sea anemones by their symbiotic shrimp

Journal

ELSEVIER

of

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JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

-I

Supply of regenerated nitrogen to sea anemones by their symbiotic shrimp Stephen Spotte Marine Sciences & Technology Center, University of Connecticut at Avery Point, Groton, CT 06340, USA

Received 8 February 1995; revised 22 August 1995; accepted 7 September

1995

Abstract

The spotted anemone shrimp, Periclimenes yucatanicus (Ives, 189 1). a frequent symbiont of the giant sea anemone, Condyhctis gigantea Weinland, 1860, excretes ammonia at the rate of 0.0393 pmol total NH,-N/(g of shrimp. min), enriching the nitrogen concentration among the anemone’s tentacles. Anemones associated recently with a shrimp demonstrated an enhanced capacity to take up external ammonia, compared with anemones not recently associated with a shrimp, and their tissues contained more zooxanthellae. Benthic invertebrates represent a potentially important and unexplored source of regenerated nitrogen on coral reefs. Keywords: Regenerated nitrogen; Ammonia; Zooxanthellae; Condylactis gigantea; Periclimenes yucatanicus

Anemone shrimp; Sea anemones;

1. Introduction

Coral reefs typically are oligotrophic environments where concentrations of inorganic nitrogen in the surrounding seawater are often < 1 ,umolll (Muscatine and Porter, 1977; D’Elia and Wiebe, 1990). Consequently, zooxanthellae (dinoflagellate endosymbionts) in the gastrodermal cells of corals, sea anemones, and other zooxanthellate invertebrates are sometimes nitrogen limited (Cook and D’Elia, 1987; Cook et al., 1988; Muscatine et al., 1989; Belda et al., 1993; Falkowski et al., 1993). Indirect confirmation comes from experiments in which extended increases in the concentration of external ammonia promote greater numbers of zooxanthellae (Hoegh-Guldberg and Smith, 1989; Muscatine et al., 1989; Dubinsky et al., 1990; Stambler et al., 1991; Stimson and Kinzie, 1991; Belda et al., 1993; Muller-Parker et al., 1994). If zooxanthellae assist the host in meeting its energy requirements (Muscatine et al., 1981; Falkowski et al., 1993), then increased cell densities might be beneficial (Meyer and Schultz, 1985b). Conversely, 0022.0981/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDf 0022-098 l(95)OO 169-7

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raising the concentration of external nitrogen for prolonged periods encourages balanced growth within a population of zooxanthellae, which lowers the translocation of fixed carbon to the host (Falkowski et al., 1993). Inorganic nutrients on coral reefs originate from several sources (D’Elia and Wiebe, 1990). One source, regenerated nitrogen excreted by inshore fishes, enhances coral growth and results in higher tissue numbers of zooxanthellae (Meyer et al., 1983; Meyer and Schultz, 1985b). Similar benefits perhaps accrue from the large communities of benthic invertebrates with which zooxanthellate organisms live in intimate contact. They represent a potentially important and unexplored source of regenerated nutrients, including nitrogen. A logical place to test this hypothesis is in the vicinity of zooxanthellate cnidarians. Zooxanthellae take up ammonia preferentially to other forms of inorganic nitrogen (Wilkerson and Muscatine, 1984). Benthic crustaceans are ammonotelic (Claybrook, 1983; Regnault, 1987) numerous on coral reefs, and often associate closely with hermatypic corals and other zooxanthellate invertebrates (Glynn, 1983; Hutchings, 1983; Odinetz-Collart and Richer de Forges, 1985; Patton, 1994; Scott, 1987; Tsuchiya et al., 1989). To make a contribution, crustacean-regenerated ammonia would have to be liberated at concentrations above background levels and then taken up by nearby cnidarians. I offer tentative evidence that these criteria are met. Model organisms were the giant sea anemone, Condylactis gigantea Weinland, 1860, and spotted anemone shrimp, Periclimenes yucatanicus (Ives, 1891), its frequent symbiont (Spotte et al., 1991). Hermatypic corals dominate other cnidarians on coral reefs, but because of its large size and ease of maintenance in the laboratory the giant anemone has been proposed as a “model polyp” for studying tropical cnidarian physiology (Kellogg and Patton, 1983).

2. Materials

and methods

2.1. General design Two experiments were designed to test predictions that ( 1) anemone shrimp excrete enough ammonia in the vicinity of their anemones to raise the concentration measurably above background levels, and (2) association with a shrimp and the added ammonia it provides is manifested in the anemone by increased numbers of zooxanthellae and the enhanced capacity of these larger populations to take up external ammonia. The first experiment involved the determination of ammonia concentrations excreted by individual shrimp maintained in sealed bottles of microfiltered seawater. Water samples were removed at regular intervals and frozen for later analysis. As a corollary, 5 in situ seawater samples over 2 days from among tentacles of anemones with and without a shrimp were collected to see whether, despite dilution effects, any supplementary ammonia was detectable. Baseline ammonia concentrations in the surrounding seawater were determined from paired samples taken in the water column - 25 cm from the anemones. The second experiment involved determining the short-term uptake of ammonia by 2 groups of anemones. Group 1 comprised anemones associated with a shrimp at the start

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of the experiments; Group 2 comprised those not associated with a shrimp. The greater uptake of ammonia by Group 1 anemones could indicate predisposition to supplemental nitrogen originating with the shrimp. Anemones were placed in sealed bottles containing microfiltered seawater spiked with ammonia. Water samples were removed at regular intervals and frozen for later analysis. As a corollary, numbers of zooxanthellae in the two groups of anemones were compared. The same shrimp and anemones were used in both experiments. Field work was conducted during March 1994 at 21”51’N 72”06’W on the northern edge of the Caicos Bank, Turks and Caicos Islands, British West Indies. Six anemones with shrimp (Group 1) and six without (Group 2) were selected haphazardly in water < 1 m deep at high tide and their locations marked with numbered floats. All shrimp were ovigerous adults. 2.2. Methods Seawater samples were collected in polypropylene syringes that were used only once. Each sample was transferred to a screw-capped polypropylene tube (Sarstedt 60.543.001 PP) through a new 0.45 pm polypropylene syringe filter (Whatman 6788-2504) that was discarded afterwards. The tubes were immersed immediately in ice, frozen within 30 min, and later transported frozen to the US. In the laboratory, samples were brought rapidly to room temperature just before analysis, and total ammonia nitrogen concentrations (total NH,-N = NH,-N + NH:-N; see Spotte and Adams, 1983) were determined in triplicate in an autoanalyzer by the phenol-hypochlorite method (Solorzano, 1969). Sargasso Sea water served as a blank; the reproducible detection limit was 0.05 pmol total NH,-N/l. 2.3. Experiment

1: Ammonia

excretion

by anemone

shrimp

In the field, shrimp were removed from their anemones 30 min prior to start of the experiment and placed in separate polycarbonate bottles (Nalge 2015-1000) containing 1 1 of microfiltered seawater (Millipore HTTP 04700, 0.40 pm polycarbonate filters). Controls consisted of six bottles containing microfiltered seawater but no shrimp. Constant temperature was maintained by tethering the bottles at the surface. Two 30 ml samples from the original common volume of seawater provided a mean time-zero ammonia concentration at 2.15 p.m.. Samples of 30 ml were withdrawn from the bottles every 40 min. The 40 min sampling intervals kept volumes removed from the total volume (120 ml from 1 1) nearly in proportion with those of the second experiment (20 min sampling intervals, 210 ml removed from 2 1, see below). Because total volumes removed accounted for < 1.5% of the starting volumes in both experiments, a correction was not made. Afterwards the shrimp were fixed in 10% formalin-seawater. In the laboratory they were blotted on paper towels and weighed. 2.4. Experiment

2: Ammonia

uptake

by sea anemones

The following day the anemones were placed in separate polycarbonate bottles (Nalge 2015-2000) containing 2 1 of microfiltered, ammonia-spiked seawater. Just before the start of the experiment, reagent-grade NH,Cl (Alpha 303384, lot E19G) was dispensed

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by pipette to each bottle from a stock solution. The addition of 250 ,ul/l was calculated to provide a minimum final concentration in the 2 1 bottles of 15 pmol total NH,-N/l, assuming an ambient concentration of zero. Transport of the water-filled bottles to the field site ( - 3 km over unpaved roads) ensured adequate mixing. The bottles were clear, allowing 87-91% of visible light to penetrate. Tethering them at the surface stabilized temperatures and guaranteed adequate illumination. Controls were six bottles containing microfiltered, ammonia-spiked seawater but no anemones. A 30 ml sample was withdrawn from each bottle to provide time-zero ammonia concentrations at 12.30 p.m.; samples were then withdrawn every 20 min through 120 min. Afterwards the anemones were fixed in 10% formalin-seawater. Tentacles of giant anemones contain most of the zooxanthellae (Kellogg and Patton, 1983). In the laboratory, each anemone’s tentacles were removed, blotted dry, and weighed. Afterwards 0.1 or 0.2 g of tissue from five tentacles were homogenized in a tissue disrupter with - 3 ml of artificial seawater. The homogenate was alternately centrifuged and the pellets resuspended in clean artificial seawater using a vortex mixer until zooxanthellae were concentrated. Four replicates were counted by loading both sides of a hemacytometer with mixed samples of resuspended pellet. Zooxanthellae per ml were multiplied by the resuspension volume to determine total numbers per g of tentacular tissue.

3. Results 3.1. Experiment

I: Ammonia

excretion

by anemone

shrimp

Ammonia increased in bottles containing shrimp (Fig. 1). The slope of the regression is 0.0393 pmol total NH,-N/(g of shrimp. min). Mean ambient ammonia in the control

pmol NHcN/g of shrimp

91 A

8

7

A A

Xme, min Fig. I. Anemone shrimp, linear least-squares regression plot of the increase in ammonia per gram of shrimp per minute from a starting concentration of 0.20 wmol total NH,-N/I. The regression line has been forced to zero. Correlation between the variables is significant (r2 = 0.90, F, 21 = 205.37, P < 0.001). The change in ammonia between bottles with and without shrimp is also significant (l-way ANOVA, F, Ih = 142.62, I’< 0.001). The shrimp ranged from 0.096-0.171 g (X=O.l37?0.023).

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1

Experiment

I (corollary),

paired comparisons of ammonia in seawater samples collected in situ (Wilcoxon’s

match pairs test, n = 30) Paired variables

T

Z

P

Group 1 vs. control

126.5

2.18

co.05

Group 2 vs. control

137.5

1.73

>0.05

Paired controls are samples collected nearby from the water column. MeansZSD

are: Group 1 = 1.7720.62,

Group 2= 1.60Z0.36,

pmol

Group I control = 1.60?0.28,

Group 2 control=

1.56?0.38

total NH,-N/I.

bottles declined (linear least-squares regression: r2 = 0.41; df 1, 22; P < 0.001). I can only speculate that microorganisms able to pass through the filters might have been responsible. The results displayed in Fig. 1, however, were unaffected because ammonia concentrations in the control bottles were subtracted when calculating ammonia excretion by the shrimp. In the corollary to this experiment, ammonia concentrations of seawater samples collected in situ were highest among the tentacles of Group 1 anemones (Table I), and these values differed significantly from the paired controls. In contrast, values of the Group 2 anemones and their paired water column values did not differ significantly. 3.2. Experiment

2: Ammonia

uptake by sea anemones

Group 1 anemones took up significantly more ammonia than those of Group 2 (Fig. 2), and the uptake by both groups differed significantly from the controls (Table 2). In some instances, plots of means against standard deviations for the repeated measures variable (time in 7 levels) showed significant correlation. However, when the analysis was performed nonparametrically (Friedman’s test), levels of significance for the main ,, pmolNY_NiL 1

Time,min Fig. 2. Sea anemones, linear least-squares regression plot of the change in ammonia with time. Just before time-zero all bottles were spiked with NH,CI (A)

Bottles

containing

anemones

associated

to a final minimum concentration of I5 pmol recently

with

a shrimp

(X= 10.67

pmol

total NH,-N/I. total NH,-N/I:

Y = ~ 0.04X + 13. IO), (B) Bottles containing anemones not associated recently with a shrimp (X = I I .80 pmol total NH,-N pmol

/l; Y = -0.03X

total NH,-N/I;

(f = 2.205 20.952).

+ 13.57), (C) Control bottles containing only ammonia-spiked

Y=O.O02X+

13.74).

Tentacular

tissue per anemone

seawater (X = 13.99

ranged from

0.446-3.736

g

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Table 2 Experiment

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measures

Effect

df effect

MS effect

df error

MS effect

F

Treatments (A, B, C) Time (O-120 min) Treatments X time

2 6 12

119.25 23.45 5.43

IS 90 90

5.61 0.64 0.64

21.24 36.89 8.55

Effects are significant at P
effect

(treatments) did not differ from post-hoc results of the parametric analysis: (A X B = 6.10, P < 0.05; A X C = 34.38, P < 0.001; B X C = 21.78, P < *NOVA x:.az 0.001). In the corollary to this experiment, mean numbers of zooxanthellae per gram of tentacular tissue were greater in Group 1 anemones (157.5 X 10” vs. 135.2 X 10h), but the difference was not significant as assessed from log-transformed zooxanthellae counts (independent t-test; t,,, = 0.52, P > 0.05).

4. Discussion

4.1. Discussion

of results

Meyer et al. (1983) reported that schools of resting grunts (Huemulon spp.) raised the ammonia concentration in the vicinity of reef corals from 0.2 to 0.9 pmol NH: /l. On an area1 basis, coral colonies (Porites ,furcutu) with grunts had significantly more tissue, higher concentrations of nitrogen, and greater numbers of zooxanthellae than colonies without grunts (Meyer and Schultz, 1985b). These results were interpreted as indirect evidence that resident fish schools are beneficial to corals (Meyer and Schultz, 1985b). When colonies of the hermatypic coral Pocilloporu dumicornis were maintained in seawater spiked with ammonia (20 pmol NH,’ /l), area1 concentrations of zooxanthellae, chlorophyll, and protein increased (Muller-Parker et al., 1994). In results reported here, a single anemone shrimp excreting ammonia at 0.0393 pmol total NH,-N/(g of shrimp. min) contributes measurably to the background concentration of - 1.60 pmol total NH,-N/l. Amounts excreted in the vicinity of a sea anemone’s tentacles are detectable in situ despite dilution effects and the small size of the shrimp. Group 1 anemones contained comparatively more zooxanthellae than those of Group 2, and the additional cells appear to have enhanced the uptake of external ammonia. Whether these effects are beneficial to the anemone has not been established. In zooxanthellate cnidarians, higher ammonia concentrations in the surrounding seawater typically produce elevated numbers of zooxanthellae (H@egh-Guldberg and

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Smith, 1989; Muscatine et al., 1989; Dubinsky et al., 1990; Stambler et al., 1991; Stimson and Kinzie, 1991; Hoegh-Guldberg, 1994; Muller-Parker et al., 1994). Although mean numbers of zooxanthellae were greater for Group 1 anemones than for Group 2, the difference was not significant. This finding was not entirely unexpected because the ammonia-spiking experiment lasted only a few hours. The null hypothesis of no significance had been based on previous studies showing substantial increases in cell numbers when zooxanthellate cnidarians are kept in ammonia-spiked seawater. These other experiments were conducted over periods of 13 days to 8 weeks. Moreover, cell number is an unreliable predictor of nutrient uptake: Raising the ambient ammonia for several days ordinarily promotes cell division, but the response can vary by species and even among conspecifics in the same treatment group (Hoegh-Guldberg and Smith, 1989). The nutritional status of the host is also important. Nutrient-satiated anemones contain more zooxanthellae than anemones that have been starved, and they take up [ “C]methylamine (an NH: analog) less rapidly (D’Elia and Cook, 1988). The nutritional status of the anemones 1 used was not assessed. However, the enhanced uptake of ammonia by those having the greatest mean numbers of zooxanthellae indicates that none was nutrient satiated. Meyer and Schultz (1985b) emphasized that although differences in zooxanthellae counts were sometimes significant, these and other effects were “subtle”. Cell number determined as a function of surface area was significant in comparisons of Porites furcata colonies associated with resident schools of grunts versus colonies without grunts. However, expressing this factor in terms of tissue mass, as in results reported here, produced differences that were not significant (Meyer and Schultz, 1985b, Table 1). If the effects of regenerated ammonia are indeed subtle, then the amount excreted by a single shrimp might be adequate to stimulate only slightly more zooxanthellae in the tissues of its sea anemone associate, and the effect could be confounded by the shrimp’s movements. Strength of site fidelity might be species specific. If so, the long-term availability of supplemental ammonia would be species dependent. Mahnken (1972) reported the spotted anemone shrimp to occur consistently on the same anemones but provided no data. In contrast, the sympatric P. pedersoni appears to change anemones frequently (Chace, 1958; Mahnken, 1972). If site fidelity is weak and the shrimp is transient, the availability of supplemental ammonia would be transient too. Some anemones at my study site were grouped closely together, even touching. One or more shrimp could easily have moved to adjacent anemones before the experiments began. Compared with a resident school of fishes, a community of sedentary invertebrates might contribute more consistently to the pool of regenerated nutrients. Because fishes and corals remain some distance apart, the concentrations of additional nutrients are diminished by daily migratory patterns of the fishes, seasonal changes in fish biomass, and current velocity (Meyer and Schultz, 1985a). A resident school of grunts excreted more than 50% of its daily allotment of nutrients within 4 h of returning to the reef after feeding in distant grassbeds (Meyer and Schultz, 1985a). These periods of fasting in the vicinity of corals alternated with abandonment of the reefs to feed result in large cyclical pulses of ammonia (Meyer and Schultz, 1985b). In contrast, benthic invertebrates live in close, mostly continuous contact with their zooxanthellate associates.

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implications

Symbiosis has proved conceptually elusive (e.g., Castro, 1988; Saffo, 1992), and discussion of any related terms should be accompanied by working definitions. Here “host” will denote the larger member (anemone) and “symbiont” the smaller member (shrimp) of a symbiotic association sensu Castro (1988) with nothing further implied. The spotted anemone shrimp is apparently an obligate symbiont of cnidarians (Spotte et al., 1991), although giant anemones without shrimps of any species are common throughout the West Indies. In what sense is the relationship symbiotic? Smith (1992) stipulated that true symbiosis requires exploitation of the symbiont by the host. The reverse situation, although permissible, is unnecessary. It seems intuitively logical that the spotted anemone shrimp should gain advantages through association with anemones (e.g., Mahnken, 1972), although none has yet been identified. Sheltering among the anemone’s tentacles might discourage predation. Perhaps the shrimp feeds on mucus produced by the anemone, or on drifting organisms or detritus captured by the tentacles. Addressing the problem will be challenging: increased fitness is untestable when a symbiont’s association with its host is obligate (Smith, 1992). Smith’s model stipulates that a demonstrable advantage must extend to the anemone, but not necessarily to the shrimp. At least one shrimp-anemone association appears to qualify. In the West Indies the anemone Bartholomea annuluta is defended by resident snapping shrimp (Alpheus armatus) against the predaceous polychaete Herrnodice carunculatu (Smith, 1977). Advantages to the snapping shrimp are not apparent. In contrast, the spotted anemone shrimp’s tiny chelae would seem unsuited for defense, and transfer of nutrients to the host might be an alternative means of meeting the criterion of Smith (1992).

Acknowledgments Supported by the Oakleigh L. Thorne Foundation. I thank P.M. Bubucis, K. Regan and L.S. Spotte for technical assistance. R.M. Clark and L.M. Mazzaro provided the tissue disrupter. A. Giblin and R.A. Bullis arranged for ammonia analyses at the Marine Biological Laboratory (Woods Hole, MA, USA). G. Adams, C.F. D’Elia, E. Koch and C. Yarish offered advice. Contribution No. 282 of the Marine Sciences & Technology Center.

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